608 
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•ome of the Causes of Decay 
of Brazil Nuts 



EDWIN ROLLIN SPENCER 

A. B. , University of Illinois, igu 
M.A., University of Illinois, iqij. 



THESIS 

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE 

OF DOCTOR OF PHILOSOPHY IN BOTANY IN THE GRADUATE 

SCHOOL OF THE UNIVERSITY OF ILLINOIS 

1920 



Some of the Causes of Decay 
of Brazil Nuts 



EDWIN ROLLIN SPENCER 

A.B., University of Illinois, igil 
M.A., University of Illinois, ig/4 



THESIS 

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE 

OF DOCTOR OF PHILOSOPHY IN BOTANY IN THE GRADUATE 

SCHOOL OF THE UNIVERSITY OF ILLINOIS 

1920 



Reprinted for private circulation from 
The Botanical Gazette, Vol. LXXII, No. 5, November 1921 



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VOLUME LXXII NUMBER 5 



THE 



Botanical Gazette 



NOVEMBER 1921 

DECAY OF BRAZIL NUTS 
Edwin Rollin Spencer 

(with plates viii-xii and three figures) 

Introduction 

Brazil nuts. Para nuts, Cream nuts, etc., are the seeds of 
Bertholletia nob His Miers and B. excelsa Humb. and Bonpl. The 
nuts are harvested in the months of January, February, and March, 
when the heavy pericarps containing the seeds fall to the ground. 
They are collected and transported from the forests to the seaports 
at a time of year when heat and moisture favor fungous growth, 
and often a cargo reaches New York 30 per cent spoiled. The 
United States Bureau of Chemistry holds that nuts are adulterated 
food if more than 15 per cent are spoiled, and requires that such 
nuts be shelled before being placed on the market. In spite of 
these measures, however, Brazil nuts reach the consumer containing 
from 10 to 25 per cent of spoiled nuts. There were 43,076,348 
pounds of Brazil and Cream nuts shipped into the United States 
in iqiq (7). It is probable that half of this amount, or 21,538,174 
pounds, were retailed in the shell. It is conservative to estimate the 
loss through spoiled nuts at 10 per cent of this amount, or 2,153,817 
pounds, an approximate money loss, at 40 cents per pound, of 
$861,526.80, which falls directly upon the consumer. Brazil nuts 
do not become rancid very readily, and for this reason they are 
not placed in cold storage during warm weather as are most other 
nuts. They are heaped in piles in supposedly dry and often very 



266 



BOTANICAL GAZETTE 



NOVEMBER 



hot rooms where, when moisture is present, fungous growth is 
favored. 

There is a wide 'difference in shell porosity of Brazil nuts, and 
a positive correlation between fungous infection and shell porosity 
has been demonstrated. Two two-pound samples of Brazil nuts 
purchased at two different grocery stores were tested for porosity 
of shell as follows. The nuts were taken one at a time and dipped 
first in 95 per cent alcohol to prevent the collection of surface 
bubbles, and then plunged two or more inches beneath the surface 



TABLE I 





Very porous 


Slightly porous 


Least porous 


Spoiled 


Percentage spoiled 


Cracked 


Spoiled 


Cracked 


Spoiled 


Cracked 


Spoiled 


in 

SAMPLE 




Sample I 






4 


4 














26 


9 














49 


6 














19 


















Sample II 






2 


I 












?8 


21 


8 














70 


5 














14 

















of hot water contained in a tall beaker. The heat-expanded air 
arose in bubbles from the pores of the shell. Table I shows the 
results obtained. The conclusion to be drawn from these data is 
that the most porous nuts are not necessarily spoiled, but readily 
become infected when conditions favor infection, while the least 
porous nuts are much less subject to infection. It is quite possible 
that so long as the water content of the nut is sufficient to support 
fungous growth, nuts with very porous shells may be entered and 
spoiled if storage temperature is favorable. Such infections prob- 
ably account for the high percentage of spoiled Brazil nuts bought 
of retailers whose wholesale patrons have scrupulously complied 
with the ruling of the Bureau of Chemistry when the nuts were 
purchased at port. 



i 9 2i] SPENCER—BRAZIL NUTS 267 

Although the use of nuts as foods and confections has recently 
become extended and general, there is but little concerning nut 
diseases in the literature, and studies of the diseases affecting the 
nuts only for the most part have been superficial. Mangin (17) 
described a "black rot" of chestnuts caused by Harziella castanae 
Bain., and found it to cause a 26 per cent loss of nuts gathered late 
in the season. Von Ivanoff (31), in studying Trichothecium 
roseum Link, found it in pure state on the kernels of Corylus avellana 
and Pinus cembra, and this, with Rand's (23) work on Coniothyrium 
caryogenium Rand, is the only serious investigation of nut parasites 
that has been made. Martz (18) reports a species of Cephalo- 
thecium on pecans in Florida. Kuhl (13) isolated Aspergillus 
flavus Mont, from Brazil nuts, but his description of both disease 
and fungus is meager. 

A few parasites of nut plants cause diseases of the nuts them- 
selves. The most serious disease of this kind is that produced by 
Pseudomonas juglandis Pierce, which, according to Smith (27), 
attacks the nuts as well as other growing parts, and "a nut in 
such cases is deformed in shape .... and the kernel .... is 
only poorly developed." Pierce (21) says that in young nuts 
the kernel is destroyed. Chestnuts are affected by Endothia 
parasitica (Murr.) A. and A. Rumbold (24) says that the hyphae 
of this parasite spread throughout the kernel. The kernel spot of 
pecan produced by Coniothyrium caryogenium Rand has incidently 
been studied by Turner (30), and by Rand (23). In addition to 
these studies, there have been some reports on storage results 
(6, 29), and McMurran (16) mentions what he considers a non- 
parasitic disease, the "black-pit" of pecan. 

The aim of the present investigation was to isolate and identify 
as many as possible of the more important fungi and bacteria 
causing deterioration of Brazil nuts. Seven distinct organisms 
have been isolated, studied, and their etiological relation to the 
nut deterioration demonstrated. The remainder of the paper com- 
prises the methods of study and descriptions of the organisms 
isolated. 

Methods 

The nuts studied were obtained from two wholesale firms in 
Chicago and from retail grocery stores in Champaign and Urbana, 



268 BOTANICAL GAZETTE [november 

Illinois, and were purchased at different times during the year 
1919-20. Each nut was superficially examined and the shell 
carefully removed by cracking it with a hammer. The diseased 
nuts were dropped, shell and all, into suitable sterilized glass dishes, 
one nut in each dish. A number was assigned to each, but only 
those diseases which were most prevalent and which presented 
the most conspicuous diagnostic features were selected for 
study. 

A preliminary examination was made of thin razor sections of 
diseased tissue mounted in water or xylol, in order to discover 
whether fungi or bacteria were present, and if so, to ascertain 
their general relation to the host tissues. If this examination 
showed any single species of organism to be predominant, isolations 
were made either by direct transfer to cornmeal agar plates, or by 
dilution plating as the case required. These isolations were from 
both exterior and interior portions of the nut, and when from the 
interior were carried out in the following manner. The nut meat 
was cut into with a flamed scalpel and carefully broken apart. 
A central portion of about 4 sq. mm. area was carefully removed 
with a flamed scalpel from one of the newly exposed surfaces, and 
discarded. In the center of the cavity thus made small pieces of 
diseased tissue were loosened with the point of the scalpel, and 
immediately carried in a sterilized loop to the surface of cornmeal 
agar plates. 

Following isolation, the next step was to determine whether 
the fungi isolated were responsible for the various diseased condi- 
tions. Two methods were used; first, pieces of mycelium or a 
few spores were placed on sterile kernels contained in sterilized 
one-inch test tubes; and second, pieces of mycelium or a few 
spores were placed upon strips of sterile nut meat, 50 /x X 5 X 10 mm., 
which were contained in tubes of sterile water, one strip on the side 
of the tube just above the surface of the. water and the other in 
the water. By the first of these methods the rotting power of the 
parasite was made evident within a few weeks by the softening of 
the whole mass. With the second method results were obtained 
more quickly by more or less complete dissolution of the very thin 
sections employed. The following media were used in the case 
of every organism. 



i 9 2i] SPENCER— BRAZIL NUTS 269 

Cornmeal agar. — This was prepared as recommended by 
Shear (26), except that the medium ready for filtration was poured 
into precipitation cones and autoclaved. After solidifying, the 
precipitated dirt at the apex of the inverted cone was removed and 
the clean agar melted and tubed. 

Brazil nut agar. — Fifty grams of Brazil nut kernels which 
were free of, or easily freed from, their inner seed coats were 
ground in a nut grinder (a Russwin no. 1 Food Cutter was used), 
and steeped for one hour at from 58 to 6o° C. in 1000 cc. of distilled 
water, counterpoised, and filtered. Fifteen grams of powdered 
or crude agar was added and the mixture boiled for ten minutes, 
counterpoised, poured into precipitation cones, and autoclaved at 
15 pounds for fifteen minutes. After solidification the agar was 
removed from the glass cone and placed on a clean sheet of paper. 
After removing the sediment the dirt-free agar was returned to the 
precipitation cone and again autoclaved. The resolidified agar 
cone was in two distinct layers, and the translucent layer was 
the one used. 

Nut plugs. — It was found possible, by flaming a scalpel after 
each stroke, to cut out nut plugs of considerable size which were 
free from contamination. The kernels from which such plugs 
were to be cut were placed in a 2 to 1000 solution of mercuric chlorid 
where they remained for thirty minutes. They were taken from 
the solution one at a time, held by one end between thumb and 
finger, and shaped by cutting away a thin layer with a sharp scalpel, 
flamed after each stroke. When the plug was finished it was cut 
off after placing it within the mouth of the reclining one-inch test 
tube. Nut plugs made in this way remained uncontaminated for 
several months. 

Nut strips. — Strips of nut meat, 50 ^11X5X10 mm., were cut 
on a microtome and preserved in absolute alcohol. When used 
they were taken from the alcohol with sterilized forceps and placed 
in sterile water in a Petri dish. From this they were removed 
with a sterilized loop and placed in test tubes containing sterile 
water, as already described. 

Autoclaved rice. — This medium was made by placing two 
or three grams of rice and twice the volume of water in test tubes 
and autoclaving. 



270 BOTANICAL GAZETTE 



NOVEMBER 



Microtome sections. — These were made in order to show the 
morbid histology in comparison with the normal histology. Because 
of the oil content of the nut, ether was found to be the best killing 
and fixing agent. The ether was replaced after three days with 
chloroform, and the ordinary schedule for imbedding with this 
reagent followed (2). After sectioning it was found that the oil 
content had not been sufficiently lessened, and that without its 
removal a distinct view of the structures could not be secured. To 
obviate the difficulty the sections were fixed to the slide, treated 
with xylol, xylol and absolute alochol, absolute alcohol, and then 
flooded four or five minutes with ether. The slide was held 
horizontally between thumb and finger, and the dissolved fats 
collected on the under side of the slide, from which they were wiped 
off before placing the slide in 95 per cent alcohol. The slide was 
kept in each of the 95, 85, and 70 per cent alcohols for about five 
minutes, and then flooded with Pianese Illb for 15 minutes (32), 
washed with distilled water, 70, 85, and acidulated 95 per cent 
alcohol, 95, 100 per cent alcohol, 100 per cent alcohol and xylol, 
xylol, and mounted in balsam. The stain shows the host tissue 
in red and the fungus in green. 

Two of the fungi produced pycnidia which were sectioned for 
study. These were taken from cultures on cornmeal agar, killed 
and fixed with chromacetic fixing fluid, and stained with Bismark 
brown, following the usual schedule for this stain. The pycnidia 
were removed from the culture on a strip of agar, usually about 
1X2X4 mm. in size, which remained intact throughout the process 
and served well in the orientation of the specimens in the imbedding 
dish. 

Freehand sections. — It was occasionally found necessary 
to stain razor sections made for the preliminary study of the dis- 
eased tissue. The sections were cut as thin as possible and placed 
in a watch glass contained in a Petri dish. The watch glass was 
then filled with ether and the Petri dish closed. When the ether 
had evaporated, 95 per cent alcohol was poured over the sections and 
allowed to stand for ten or fifteen minutes. This was followed 
with 85, 70, and 50 per cent alcohols for five minutes each. The 
sections were then transferred to slides prepared with albumen 



i 9 2i] SPENCER—BRAZIL NUTS 271 

fixative, flooded with water, and allowed to stand over night. 
They were stained with Pianese Illb, or with jod griin-erythrosin 
in the following manner. They were placed in 95 per cent alcohol 
for five minutes, flooded with jod griin (1 per cent solution in 95 
per cent alcohol) for thirty minutes, washed with 95 per cent 
alcohol, then absolute alcohol, flooded with 1 per cent solution of 
erythrosin in clove oil for forty-five minutes, washed with absolute 
alcohol, cleared with carbol-turpentine clearer, and finally washed 
in xylol and mounted in balsam. This proved to be the most 
satisfactory of any method tried for staining mycelium in the 
tissue. 

Proteolytic enzymes. — The Brazil nut agar serves well to 
show the presence or absence of certain extracellular proteolytic 
enzymes. The proteid precipitates to which the opacity of the 
agar is due are digested by the enzymes, and a transparent halo, 
which enlarges as the thallus or colony enlarges, surrounds the 
growth. All the organisms studied were tested for the presence of 
these enzymes. The enzymes were precipitated from cornmeal 
broth in which an Actinomyces or a Bacillus, the two most active 
enzyme producers, was grown. The broth was poured into Pior- 
kowski culture flasks to a depth of 0.25 inch, about 200 cc. being 
required for each flask, and inoculated. After ten days the culture 
was filtered through paper and enough 95 per cent alcohol added 
to the filtrate to make 80 per cent alcohol. Three days later a 
fluffy white precipitate had collected at the bottom of the precipita- 
tion cones, and the excess alcohol was siphoned off (12). From 
25 to 50 cc. of absolute alcohol was added to the precipitate and 
immediately filtered. Before the precipitate had become dry 
it was again washed with 50 cc. of absolute alcohol, and while still 
moist was removed to a desiccator containing sulphuric acid, and 
allowed to remain there for two days. The hard, gray material 
was then scraped from the paper to be redissolved in sterile water 
when used. 

Morphology of Brazil nut. — The kernel of the Brazil nut, 
as it is ordinarily removed on cracking the shell, is covered with 
a thin, dry coat which may be quite loose or may adhere very 
tenaciously. The embryo within, principally radicle, is completely 



272 



BOTANICAL GAZETTE [November 



enveloped with a layer of endosperm 40 to 50 /j, in thickness, and 
as reported by Young (35) is "plainly differentiated into cortical 
and medullary tissues separated by a layer of procambium along 
which rudimentary vascular bundles are arranged at intervals." 
There is a single, somewhat irregular layer of epidermal cells just 
beneath the endosperm, and within 5 mm. of the distal end are the 
two very minute, unequal cotyledons which "measure about 750 
by 175 m" ( j > 2 5)- The cortical and medullary cells are similar 
in shape and size, and are largely rilled with oil and proteid bodies. 
The endosperm cells, arranged with the long diameter at right 
angles to a median plane, are especially rich in proteids (28) . The 
procambium cells with the long diameter at right angles to that of 
the endosperm cells contain few or no proteid grains (fig. 1). 

The outer seed coat or shell is made up of two layers ; the outer 
with its crinkled surface is light brown and softer in texture than 
the inner layer, which is dark brown and has a glazed inner surface. 
In the angles of the shell this layer seems to be of two layers which 
divide, leaving spaces filled with still another tissue that is lighter 
in color and softer in texture than the outer of the shell layers. In 
the micropylar angles of the seed is a narrow cavity. Such cavities 
are termed by Berg (i) the "loculi spurii in testa," and extend the 
entire length of the shell. This open channel probably serves as 
the usual entrance of the parasites of the nut (fig. 1). 

The tissues of the seed, taken in order, beginning with the shell 
are: (1) outer seed coat in two distinct layers, with a softer tissue 
filling the triangularly prismatic corners; (2) the thin inner seed 
coat which may or may not adhere to the kernel; (3) the endosperm 
layer, two cells thick; (4) epidermis, a single-celled layer; (5) cor- 
tex, of large storage cells; (6) procambium layer, generally three 
cells thick; (7) the medullary tissue, of large storage cells. 

Diseases of the Brazil nut 

1. Black crust 

General description. — Fully 5 per cent of all diseased Brazil 
nuts are affected with black crust, but there is no external indica- 
tion of their condition, since the shells are normal in color and the 



1921J 



SPENCER— BRAZIL NUTS 



273 



weight is the same as that of sound nuts. When the shell is 
removed the kernel presents a dull black appearance which, if the 
whole nut is affected, reminds one of a large sclerotium. A cross- 
section of the diseased kernel shows the blackened portion to consist 
of a thin layer, 100-250 /x in thickness, apparently having no 
connection with the tissues beneath, which, aside from their light 
brown color and their pronounced nut odor, appear to be normal 




Fig. 



Fig. 2 



Figs, i, 2. — Fig. 1, Diagrammatic drawings of cross and longitudinal sections 
of Brazil nut: a and a 1 , locule in testa; b, endosperm layer; c, procambium layer; 
d, medullary tissue; e, outer layer of outer seed coat;/, inner layer of outer seed coat; 
g, soft tissue filling corners of shell; h, inner seed coat; fig. 2, pycnidium with imma- 
ture spores, Pellioniella macros pora n. sp.; X500. 

(figs. 34-36) . The diseased nut meats are frequently found covered 
with various fungi, chiefly Penicillium or Aspergillus, with black 
crust under the mold. A study of microtome sections shows that 
the mycelium is in the endosperm layer, the affected cells of which 
are hypertrophied (figs. 34, 35). The cortical cells of the radicle 
immediately beneath are not parasitized, but their contents are 
markedly changed. The proteid grains are almost or quite lacking 
in the epidermal and outer layers of the normal cortex, while in 
diseased nuts there is a superabundance of small proteid grains in 



274 BOTANICAL GAZETTE [November 

this region (figs. 34, 35). As it is possible to find nuts seemingly 
free from any other organism, the black crust fungus is easily 
isolated. Small pieces of tissue taken aseptically from immediately 
below the crust on direct plating gave pure cultures. 

Morphology. — The mycelium on cornmeal agar is of two 
kinds, that made up of cells which are longer than wide, and that 
with cells either nearly globular or wider than they are long (figs. 18, 
19). The long-celled type predominates, both in the aerial and 
the submerged mycelium, except near pycnidia, where the shorter 
cells are most in evidence. The long cells are 14-32 ]x in length 
by 3 . 5-14 m in width, the short ones measuring 10-18 ju. in diameter. 
Both types are thick-walled and black when mature, and both 
have granular contents (figs. 19, 20). In autoclaved rice and in 
the black crust of diseased nuts the hyphal cell is transformed 
until the hyphae suggest chains of conidia (figs. 14, 20). These 
cells are black, 10-15X5-8/1 in size, and contain one or two 
guttulae. They readily break away from the hyphae and function 
as spores. 

Pycnidia are produced sparingly, and only along the border of a 
thallus where it comes in contact with another thallus, either of the 
same or of some other species. No pycnidia were found on dis- 
eased nuts or on any of the cultures except those on cornmeal agar 
plates. They are black, smooth, globose-conical, beaked, and 
150—350 \x in diameter. The beak is 100-250 \x in length (figs. 2, 21). 

The spores are borne at the base of the pycnidial cavity on 
short, hyaline, often septate conidiophores which are interspersed 
with narrow strap-shaped, hyaline, continuous paraphyses that 
are from one to six times as long as the conidiophores, the conidio- 
phores being 5-14 m in length by 3-5 n in width (fig. 13). The 
spores are at first hyaline, unicellular, 26-36 X 14-20 \i in size and 
irregular in shape, but with maturity they become sooty black, 
striated, uniseptate, regular in shape and uniform in size, being 

28X14 M (fig- 15)- 

Culture characters. — On cornmeal agar plates the fungus 
grows at the rate of 0.5-0.7 mm. per day at room temperature. 
The thallus is at first milk white, and the margin of it remains 
uncolored so long as it is increasing in size. After five or six days 



iq2i] SPENCER— BRAZIL NUTS 275 

the central portion becomes green, and a few days later turns sooty 
black. The thallus then is made up of three concentric rings, the 
outer white, the next green, and the innermost black (fig. n). 
As the thallus ages it shows marked zonation, and becomes entirely 
black when growth ceases. Aerial mycelium is produced on all 
parts of the thallus, but is most luxuriant in the central area. On 
Brazil nut agar the growth is very much slower than on cornmeal 
agar, usually 0.3 mm. per day at room temperature, and the en- 
tire thallus remains hyaline. On nut plugs the growth was very- 
weak, but a crust similar to that of naturally diseased nuts was 
formed after three months. On autoclaved rice the growth was 
vigorous, and several characteristic color reactions were noted 
(fig. 8) . Eight days after inoculation : aerial mycelium snow white 
with line of Antique Green 1 below; rice grains in contact with 
glass, white bordered with Cerulian Blue; all interstices with 
greenish tints. After fifteen days: aerial mycelium white with 
lower border line Prussian Blue, almost black; contacts of grains 
with glass white bordered with Prussian Blue; interstices purple 
tinged. 

The hyaline immature spores as well as the black mature ones 
germinated readily. The immature spores occasionally germinated 
in ten minutes after planting, while more than an hour is required 
for the germination of the mature spores, but the germ tubes of the 
mature ones soon outstrip those of the immature (figs. 16, 17). 
There is no change in either spore, except a slight swelling in 
germination, the immature spore remaining unicellular. This phe- 
nomenon of the germination of immature as well as mature spores 
has been pictured by Higgins (ii) for a related species. 

Taxonomy. — The morphological characters of the fungus are 
those of PellionieUa Sacc, but according to Saccardo (25) there 
is but one species in the genus, P. deformans Penz. and Sacc, 
whose spore measurements are a little more than half those of the 
Brazil nut parasite. The fungus, therefore, is given the name 
PellionieUa macros por a. 

1 The nomenclature used in describing colors throughout these investigations is 
that given in Robert Ridgeway's Color standards and color nomenclature, pub- 
lished by the author, Washington, D.C. 1912. 



276 BOTANICAL GAZETTE [November 

Pellioniella macrospora, n. sp. — Pycnidia sparse, smooth, 
carbonaceous, globose-conical, beaked, 150-350 p in diameter, 
beak 100-250 fx in length. Conidiophores at base of pycnidial 
cavity, hyaline, often septate, 5-15X3-5 m- Paraphyses hyaline, 
strap-shaped, continuous, 5-50 p. Immature conidia hyaline, 
unicellular, irregular, 26-36X14-20 n; mature conidia sooty black, 
striated, uniseptate, regular, 28X14 ju- 

Habitat. — Parasitic on endosperm of seed of Bertholletia 
nobilis Miers and B. excelsa Humb. and Bonpl. 

2. White mold 

General description. — White mold is not so common as 
black crust, and is probably responsible for less than 1 per cent of 
the Brazil nut decay, but it is a real factor in this loss. The 
diseased nut is normal in external appearance, but is below normal 
in weight. When cracked, the white, fluffy mycelium is seen to 
cover the entire kernel, but soon after exposure the hyphae collapse 
and the yellowed endosperm becomes visible through the mycelial 
mass. A pronounced musty odor arises from the newly shelled 
nut, but the taste of the diseased meat has nothing to distinguish 
it. A cross-section of the nut kernel shows three typical features 
of the disease: (1) the white moldy covering; (2) the endosperm 
layer, sulphur-yellow in color and more than twice as thick as in 
the normaj nut; and (3) irregular cracks and cavities in the 
radicle, all filled with white mycelium and spores. The mycelium 
penetrates to the center of the radicle. The hyphae in the tissue 
are very tenuous, less than 2 jjl in diameter, and are usually so 
closely associated with the cell walls of the host tissue as to make a 
study of them in situ very difficult, but the cell walls of the diseased 
nut are penetrated by them. The fungus was isolated as described, 
but spore dilutions made by touching a sterile loop to the mycelial 
mass in the internal check of the kernel gave pure cultures also. 

Morphology. — Mycelium taken from the nut, from nut plugs, 
and from other media was uniform in character. The following 
description is of mycelium taken from cornmeal agar plates. The 
cells measure 20-70X3.5-10.5^, and are hyaline with granular 
contents and guttulae. Anastomosis of cells is of frequent occur- 



IQ2l] 



SPENCER— BRAZIL NUTS 



277 



rence, especially in the submerged hyphae, while in the aerial 
mycelium simple loops and coils are common (fig. 24). The hyphae 
are unconstricted at the septa, and unbranched cells are of quite 
uniform size throughout their length. Cells bearing branches 
are swollen at the points from which the branches arise. 




ft 00 /\n. ir.HS en 

Fig. 3. — Development of spore mass of Cephalosporin))! berthoUetianum n. sp.; 
dotted line shows size of water drop surrounding mass; X 100. 

The conidiophores, 50-90 jx in length, are in most instances 
simple branches from either a principal filament or from its branches. 
They are spindle-like, with rounded tips from which the spores 
are cut off (fig. 3). The single-celled, oblong-elliptical, hyaline 
conidia are 8-12X3.5-54 ju. in size, and contain two guttulae 
(fig. 23). They collect, as they are produced, in a spherical mass 
at the end of the conidiophore. A drop of water surrounds the 
spore mass after the third spore arrives (fig. 3). 



278 BOTANICAL GAZETTE [November 

Culture characters. — On cornmeal agar the fungus grows 
rather rapidly, 0.5-1.0 cm. daily at room temperature. The thal- 
lus is very regular and is distinctly zonated after reaching a 
diameter of 4 or 5 cm. There is a dense growth of white aerial 
mycelium on the older portions of the thallus. On Brazil nut 
agar the characters are as previously stated, except that the 
growth is a little more rapid, and a halo 3 mm. wide, due to the 
digestion of the solid proteids, surrounds the thallus. On nut 
plugs the fungus grows luxuriantly and destroys the nut meat 
without giving off any appreciable odor. A large amount of fluffy 
mycelium is the characteristic feature of its growth on nut plugs 
as it is on the nut in the shell. On autoclaved rice the growth is 
vigorous and a pink tinge appears in the medium after two days. 
After ten days four colors are distinguished; where the rice is in 
contact with the glass in the older portions, Ochraceous Buff, in the 
younger, Venetian Pink; interstices between the grains are filled 
with mycelium through which a Jasper Pink color shows, in the 
older portions ; in the younger portions it is Light Hortense Violet. 

The nut strip above the water is soon covered with the dense 
mycelium, and is appreciably shrunken within five days. The 
strip in the water remains intact, but the water is soon filled with 
the mycelium which makes its way upward from the strip at the 
bottom. In hanging drop the spores germinate with a single 
germ tube, which in the most vigorous, at room temperature, may 
attain the length of the spore in two hours after planting. Spore 
production in hanging drop at room temperature proceeds at the 
rate of about one spore per hour per conidiophore. The conidio- 
phore lengthens and increases in diameter as the conidia are cut 
off at the end. The process is very like spore production of Tricho- 
thecium as described by Lindau (14). 

Taxonomy. — The fungus evidently belongs to Cephalosporium, 
but none of the species of this genus as reported by Saccardo 
(25) has characters sufficiently like the Brazil nut parasite to permit 
it to be classified as one of them. C. fructigenum McAlp. (15) has 
spores of almost identical shape, size, and appearance, but it has 
knobbed conidiophores and oblong spore masses which are not pres- 
ent in this species, which therefore is described as new. 



1921] SPENCER— BRAZIL NUTS 279 

Cephalosporium bertholletianum, n. sp. — Conidiophores hya- 
line, simple or dichotomously branched, 50-90 ix long, 2- to 4-septate; 
spore mass globular; conidia hyaline, unicellular, oblong-elliptical, 
guttulate, 6-12X3.5-5 m, ends obtuse. 

Habitat. — On radicle of seed of Bertholletia nobilis Miers and 
B. excelsa Humb. and Bonpl., causing decay. 

3. Dry rot 

General description. — -The shell of the nut affected by dry 
rot is mottled, but of somewhat lighter shade in its darkest areas 
than the shells of normal nuts, and the weight is much below normal. 
The cracked shell appears to be filled with a kernel which adheres 
more closely to the shell than is usual, but which is so similar in 
color and general appearance to that of sound nut kernels that it 
might easily pass casual observation as such, although in reality 
it is merely a mass of mycelium. Small pieces of mycelium taken 
from this mass swell to approximately twice their size when placed 
in water. Under the microscope the hyphae were seen to be 
irregularly branched and septate. No conidiophores were seen, 
but what appeared to be unicellular elongated conidia of greatly 
varying length were occasionally found. 

Morphology. — The hyphae which make up both the aerial 
and the submerged mycelium are irregularly branched, and more 
or less constricted at the septa. The cells are 14-90 fi long by 
3.5-1 1 jx wide, hyaline with granular contents and guttulae (fig. 44). 
Anastomosis frequently occurs, especially in older thalli, when 
spores falling on the medium germinate, producing a tube which 
unites with the cell of an older hypha, another germ tube, or an- 
other spore (figs. 29, 48). 

The simple conidiophores are borne at any place along the 
hyphal strand, seldom more than two being produced by a single 
cell. Branched conidiophores are rare. Thalli resulting from 
direct planting of mycelium taken from the diseased nut produce 
but few conidiophores, and rarely more than single-celled conidia. 
Conidia from transferred cultures are from 1- to 8-celled, sub- 
cylindrical, slightly sickle-shaped, without pedicel, and conical 
at base (fig. 42). 



280 BOTANICAL GAZETTE [November 

No perithecia were found, but sclerotia were formed on auto- 
claved rice. These are dark gray and 500-1000 fx in diameter. 
Terminal chlamydospores are produced on 60-day old cultures. 
They are globular or oblong, with an average mean diameter of 14 /z, 
and with a scarcely perceptible yellow tinge (fig. 45). 

Culture characters. — Pure cultures are easily obtained by 
directly planting pieces of mycelium taken from the innermost 
portion of the mycelial mass. On cornmeal agar the rate of 
growth averages 0.5 cm. daily at room temperature. The thallus 
is arachnoid and regularly zonated, the zones averaging 0.5 cm. 
in width. Aerial mycelium covers the entire thallus but is most 
luxuriant in the central area, and there it is tufted. On Brazil nut 
agar the growth is more dense and a little more rapid than on corn- 
meal agar, its rate being from 0.7 to 1.0 /j, at room temperature. 
An extra-cellular, proteolytic enzyme is secreted, causing a halo 
of 3-5 mm. in width in the medium surrounding the thallus. The 
aerial mycelium is more luxuriant on this agar than on the other. 

The fungus grows vigorously on nut plugs, so that in a few days 
the plugs are enveloped with the snow-white mycelium, while a 
putrid odor is exhaled. After two or three months the plugs are 
completely reduced and only a mycelial mass remains. There is 
no color change on autoclaved rice until it shrinks away from the 
tube, when it is Maize Yellow. The fluffy, white, aerial mycelium 
surmounts the rice column and covers its sides as the growth 
proceeds downward. Apparently complete destruction of the 
rice is accomplished within two months. On carrot plugs the 
growth is not so rapid as on other media, but is marked by an 
abundance of white aerial mycelium. It is without color change. 
On the strip of nut meat above the water the growth is vigorous, 
and if the water surface remains near enough to the strip it is 
destroyed within eight or ten days. The strip in the water often 
appeared to be intact when it was not, the mycelium retaining the 
outline. It was probably destroyed as soon as the strip above. 

In hanging drop the conidia begin germinating after three or 
four hours at room temperature, but many of them require twenty- 
four hours or more. Seldom more than two cells of a spore germi- 
nate, but frequently one cell produces two germ tubes (fig. 46). It 
often happens that spores are united by a short germ tube. Occa- 



19 2 1] SPENCER— BRAZIL NUTS 281 

sionally four or five conidia are connected in this way (fig. 47), 
resulting, as is clearly shown by drop cultures, from germination 
succeeded by anastomosis (fig. 48). In all cultures the dense 
mycelium collects and retains water enough to germinate the 1- and 
2-celled spores, and their genu tubes anastomose readily with the 
first cells, conidial or hyphal, with which they come in contact. 
It is often difficult to distinguish between conidiophores bearing 
conidia and conidia anastomosed to hyphal cells with a short germ 
tube (fig. 44). 

Taxonomy. — The fungus is a species of Fusarium which, 
according to Wollenweber's (34) scheme of classification, belongs 
to the section Eupionnotes: chlamydospores present; perithecia 
unknown; conidia subcylindrical, sickle-shaped; base without 
pedicel, conical; terminal chlamydospores. 

4. Aspergillus decay 

General description. — Brazil nuts attacked by Aspergillus 
may give no external indication of their internal condition except 
in the most advanced stages of the disease, when the weight of the 
nut is appreciably lowered. The kernel shrinks, often cracks 
open, and is always covered with a mass of dark brown spores. 
The odor of the diseased nut is strongly rancid with a putrid taint; 
the taste is at first sour, later very bitter. Occasionally nuts that 
are merely discolored have this same taste. Kuhl (13) states that 
Brazil nuts affected with Aspergillus flavus Mont, are poisonous, 
and that the discoloration caused by this fungus is so slight that it 
does not prevent their being eaten. Both his observations and my 
own indicate that the disease, although present, may often escape 
notice, and that it is really far more prevalent than it appears to 
be under superficial examination. Nuts in advanced stages of the 
disease, however, occur less frequently than black crust. The 
mycelium of the fungus penetrates the tissues to the center of the 
nut, and when there is a central locule, appears as a white mold on 
the walls of the locule. When the diseased kernels crack open, a 
mass of spores tills the locular space. 

Morphology. — The mycelium consists of irregularly branched 
hyphae which are slightly constricted at the septa (fig. 31). The 
cells are 20-65 X3.5-11 M> with granular contents of a faint greenish 



282 BOTANICAL GAZETTE [November 

tint. Conidiophores varying from ten to several hundred microns 
in length arise at irregular intervals from the hyphae. The shortest 
of these have little or no filamentous part, but consist merely of 
the head and sterigmata (fig. 28). Sterigmata are also borne singly 
and in groups of from two to four on the hyphal cells (fig. 27). 
The heads of the conidiophores measure 10-20 jx in diameter, and 
the sterigmata, from two to many per head, are 10-12 X 5-7 ju- 
The globular, echinulated conidia are of different shades of 
yellow, and 5-10 ju in diameter, but the predominant size is 7 \x 
(fig. 29). 

Culture characters. — On cornmeal agar the rate of growth 
varies from 0.3 to 1.0 mm. daily at room temperature, and after 
forty-eight hours the central portion of the thallus shows the 
forming spore clusters in Light-Buff. The spore masses become 
darker with age until Lemon-Chrome is finally reached. On Brazil 
nut agar the growth is very similar to that on cornmeal agar, but 
with a halo 1 to 2 mm. in width, showing the presence of an extra- 
cellular, proteolytic enzyme surrounding the thallus. The color 
of the spore mass at maturity is from Orange-Cinnamon to Mikado- 
Brown. On nut plugs the growth is rapid, and a gas with the 
odor of carbon bisulphide is evident. The color of the spore mass 
is Primrose-Yellow at first, Honey-Yellow to Tawny-Olive at 
maturity. At the end of two or three months all that remains of 
the nut plug is a mass of partially disintegrated cell walls in a mass 
of mycelium. 

The growth on autoclaved rice is vigorous, with spore masses 
forming within forty-eight hours. There is little change in the 
color of the medium except for the development of a slight greenish- 
yellow tint below the spore mass. The color of the spore mass 
changes from Oil- Yellow to Orange-Cetrine. The odor of a 30-day 
old culture is very like that of cider vinegar. The nut strip above 
the water is entirely covered with spore masses within three days, 
but only about one-fourth of it is destroyed before it becomes too 
dry to support the fungus. A luxuriant growth of mycelium arises 
from the strip in the water, and usually the strip is destroyed before 
fifteen days. 



IQ2l] 



SPENCER— BRAZIL NUTS 283 



Taxonomy. — A culture of this species was sent to Charles 
Thom, and the following excerpt is taken from his reply dated 
January 29, 1920: 

The organism belongs to the general group in which we are trying to 
separate three lines, the Aspergillus oryzae-flavus line, the Aspergillus wentii 
section, and the one which has been designated by Kita as Aspergillus tamari. 
This one, from the examination today, would appear to belong to the section 
containing A. tamari. Whether it is safe to identify it under an existing 
name or not would be doubtful. 

5. Bacterial decay 
General description and morphological characters. — 
When Brazil nuts are affected by this bacterial decay, the shell 
is black and greasy, and usually exhales a rancid odor. When the 
shell is cracked open the remains of the kernel are found as a 
white mass which ordinarily fills only a small portion of the shell 
cavity. Microscopic examination of fragments of the refuse shows 
numerous bacterial spores, but usually no vegetative forms and no 
fungi. When dilution plates were made from the decayed residue, 
one spore-bearing organism largely predominated. 

The vegetative cells of the organisms in cornmeal broth are 
rod-shaped, rounded at the ends, vigorously motile, and usually 
single but often in chains of from two to six individuals. The rods 
measure 2.5-5.0 mXo.8-i. 2 /x- Spores are formed within forty- 
eight hours in one end of the vegetative cells. When the cells are 
stained by Loeffler's method, the organism is found to have 
numerous long, peritrichiate flagella; stained with Loeffler's 
methylene blue the protoplasm is seen to be granular with from two 
to four darkened patches which are unevenly distributed, usually 
giving a banded effect, although often the bands are oblique as well 
as horizontal (fig. 38) . The organism stains readily with methylene 
blue, Gentian violet, and carbol-fuchsin, but it is Gram negative. 

When sterile nut plugs were inoculated with the bacillus from 
pure culture, they were reduced in about fifteen days to an oily 
mass which, in all essential characters, was like the remains of the 
nut kernels in the natural cases of nut decay. Dilution plates 
made from nut meats that had decayed, following pure culture 



284 BOTANICAL GAZETTE [November 

inoculation, showed only one type of colony, and this proved to 
consist of the organism with which the plugs had been inoculated. 
The organism had no appreciable effect on the nut strip above the 
water, and the strip in the water was only very slowly decomposed, 
but strips in cornmeal bouillon were completely destroyed within 
ten to fifteen days. The organism grows best in the presence of 
air, as the colonies on all plated media and stab culture show, but 
deep lenticular colonies (fig. 40), and colonies next to the glass 
(fig. 39) in agar plates, as well as the faint line of growth along 
the stab, indicate that it is a facultative anaerobe. 

While none of the usual tests for particular enzymes was 
made, the reactions in different culture media indicate the produc- 
tion of diastase, invertase, rennet, and pepsin. In Brazil nut agar 
plates there is formed a transparent halo about the colony, and as 
the opacity of the agar is due to the presence of solid proteid matter 
(20), the halo results from the digesting of these proteids. There 
is an abundant secretion of the protease which makes the halo, 
as the diameter of the transparent area is from two to three times 
that of the colony itself. This enzyme was precipitated as already 
described, and drops of a water solution of the dried precipitate 
placed on Brazil nut agar plates. A transparent area as large as 
the drop of solution was formed in a plate 2 mm. thick in from two 
to three hours. 

The organism seems to be an undescribed one, and a complete 
description of it will be given in a separate paper. 

6. Actinomyces decay 

General description and morphology. — Empty shells that 
are intact and still retain their normal color are occasionally found 
among Brazil nuts. When these shells are cracked open a char- 
acteristic musty odor is evident, and the inner shell wall is seen 
to be covered with pinkish velvety pustules that are from one to 
several millimeters in diameter. Water mounts of pieces of a 
pustule show tenuous, mycelial-like strands, or chains of spores 
which readily stain with carbol-fuchsin. The filaments are not 
long but branch, and the mass is so bound together by the branches 
that it is quite impossible to separate entire filaments from the mass. 



i 9 2i] SPENCER— BRAZIL NUTS 285 

The filaments are never entirely straight nor yet very crooked, and 
chains of spores are usually contained in the free ends (fig. 37). 
No spirals were found on any of the media. The diameter of the 
filaments varies from 1.0 to 1.3 jx, and the oblong spores measure 
1.6X0.8 fX. 

The germination of spores was studied with an oil immersion 
lens, in a hanging drop prepared as follows. A thin film of synthetic 
agar was spread on a thin cover-glass, and a loop full of a dilute 
spore suspension placed on the agar film. This was inverted over 
a dry Van Tieghem cell. The water soon evaporated, leaving the 
spores in contact with the agar, where their germination was 
easily studied and camera lucida drawings made. According to 
Drechsler (9), Actinomyces spores produce from one to four 
germ tubes, "the approximate number being more or less char- 
acteristic of the species." This species produces one and two 
germ tubes which often branch directly on leaving the conidium 

(% 36). 

The organism was studied in the manner suggested by Conn 
(3) and Waksman (33), and the media were made in accordance 
with directions given by Waksman (33). The following culture 
characters were noted : 

Cultural characters. — i. Synthetic agar: room tempera- 
ture, after ten days: growth densely compact but thalli small, at 
first white, but after ten days Pale Pinkish Buff; aerial mycelium 
white and dense; soluble pigment none. 

2. Calcium malate-g]ycerin agar: growth spreading and not 
zonated, bordered by submerged mycelial bands of varying width, 
pearl white; aerial mycelium short, loose, and pearl white; soluble 
pigment. 

3. Glucose agar: growth luxuriant, color same as in synthetic 
agar, thallus conspicuously zonated; aerial mycelium white to 
Pale Pinkish Buff, powdery; soluble pigment none. 

4. Glycerin agar: growth densely compact, not zonated, Pale 
Pinkish Buff; aerial mycelium powdery, white; soluble pigment 
none. 

5. Brazil nut agar: growth rapid, densely compact with wide 
margin of submerged mycelium, white to Pale Pinkish Buff; 



286 BOTANICAL GAZETTE 



NOVEMBER 



aerial mycelium dense, white; soluble pigment none; enzymatic 
zone three to four times the diameter of thallus. 

6. Cornmeal agar: growth dense but zonated, Pale Pinkish 
Buff; aerial mycelium powdery; soluble pigment none. 

7. Egg albumin agar: growth thin, conspicuously zonated, 
Pale Pinkish Buff; aerial mycelium powdery, Pale Pinkish Buff; 
soluble pigment none. 

8. Nut plugs: growth vigorous, Pale Pinkish Buff; aerial 
mycelium powdery, white; medium not completely destroyed, 
but much shrunken and blackened. 

9. Autoclaved rice: growth vigorous, Pale Pinkish Buff; aerial 
mycelium 2 cm. ; almost completely destroyed in sixty days. 

10. Potato plugs: growth vigorous, crumpled, Pale Pinkish 
Buff; aerial mycelium abundant, at first white, later Pale Pinkish 
Buff; medium with no change in color, much reduced in size in 
two months. 

11. Carrot plugs: growth at first slow, appearing after five 
days, crumpled and dense, Pale Pinkish Buff; aerial mycelium 
powdery-, at first white, later Pale Pinkish Buff; medium darkened 
near the growth, no change in color in other regions, much shrunken. 

12. Brazil nut bouillon: growth surface pellicle, snow-white; 
aerial mycelium white, powdery; medium somewhat clarified. 

13. Nut strips: growth slight on strip above water, and none on 
strip in water; no growth on surface of water. 

Biochemical features. — The proteolytic enzyme which makes 
the halo in Brazil nut agar plates was the only one studied, but the 
growth reactions in different media were taken to indicate the 
probable production of several other enzymes, diastase and inver- 
tase especially. The proteolytic enzyme was isolated by precipita- 
tion, as previously described, and its proteolytic power tested by 
placing drops of a water solution of the dried precipitate on Brazil 
nut agar plates. Transparent areas the size of the drops developed 
in from two to three hours, depending upon the thickness of the 
agar plates. 

Taxonomy. — The organism is an Actinomyces which, according 
to Waksman's key, belongs in division B, "no soluble pigment 
produced on gelatin or other protein media," and in section /, 



1921] SPENCER— BRAZIL NUTS 287 

"species strongly proteolytic; gelatin liquefied rapidly, milk 
clotted and peptonized rapidly." No species given in this division 
and section, however, has the characteristics of the one found in 
Brazil nut shells. It is therefore given the name of Actinomyces 
brasiliensis. 

Actinomyces brasiliensis, n. sp. — Straight, branched hyphae 
1. 0-1.3 /x in diameter; spores borne in chains in free ends of hyphae, 
oblong, 1.6X0.8 ju; growth Pale Pinkish Buff on all agars except 
calcium malate-glycerin, on which it is white ; zonated on glucose, 
cornmeal, and egg albumin agars; aerial mycelium on all media, 
white to Pale Pinkish Buff; no soluble pigment formed. 

Habitat. — Parasitic on kernels of Brazil nuts. 

7. Phomopsis decay 

General description. — -Only one nut was found affected 
with Phomopsis decay, but because of its striking diagnostic 
features the fungus was isolated and studied. There was no 
external indication of the diseased condition, but the kernel of the 
nut was rich brown, with a few black specks near one end. The 
odor of the nut was pleasant and the taste agreeable. Stained 
hand sections showed that the mycelium of the fungus had pene- 
trated into the radicle to considerable depth. 

Morphology. — The mycelium was tenuous, septate, and at # 
first hyaline, but soon became brown or smoke colored. According 
to Diedicke (8), the form of the pycnidia is greatly varied. In the 
Brazil nut species several of the forms pictured by Diedicke were 
observed, but the one most commonly met with was mammiform, 
with a wartlike protuberance. The irregular pycnidial cavity so 
common to the genus was frequently observed, but a regular* 
cavity was the rule. Two forms of spores were present in all 
pycnidia examined (fig. 50), and as is customary, the Phoma type 
will be designated as A, the filamentous as B spores. The B 
form did not germinate in hanging drop, a fact supporting the 
statement made by Grove (10) that these may or may not be 
spores. When they fail to germinate they are probably what 
Saccardo (25) took them to be, conidiophores, which according 
to Grove "become more curved than when in situ." The A 



288 BOTANICAL GAZETTE [November 

spores are oblong-elliptical, hyaline, guttulate, and measure 
5-7X1.7-3.5 ju. The B spores are filiform, usually hook-shaped, 
hyaline, continuous, and measure from 17-24.5X2-3.5 /x- 

Culture characters. — The fungus grew well on all media 
used. On cornmeal agar the thallus was circular and without 
zonations. A loose aerial mycelium covered the entire thallus, 
and numerous pycnidia varying in size were scattered over the 
surface of the plate. The pycnidia appeared simultaneously with 
the brown color, which was usually noticed after five or six days. 
On Brazil nut agar the growth was similar to that on cornmeal 
agar. The clear halo formed in the agar plate barely exceeded 
the size of the thallus. On autoclaved rice a brown or smoky color, 
due to the mycelial growth, was noticeable, but no color change 
occurred in the medium. Nut plugs were soon covered with a 
brown mycelium which later became almost black. The surface 
was soon covered with black, wartlike pycnidia, and the entire 
mass when cut through suggested a dried sponge. The odor was 
similar to that of very rancid nuts. The fungus made no growth 
on nut strips above the water, but a dense mass of mycelium, filled 
with black strands, developed on the strips in the water. These 
strips retained their form, but the cessation of mycelial growth, 
which occurred between ten and fifteen days after inoculation, 
marked the time of nutrient exhaustion. 

Taxonomy. — The fungus is a typical Phomopsis which 
approaches P. aucubicola Grove more nearly than any other 
described species. A spores are shorter, and this, coupled with the 
fact that it occurs on an unrelated host, necessitates describing it 
as new. 

B Phomopsis bertholletianum, n. sp. — Pycnidium dark brown, 
mammiform, with wartlike protuberance, irregular in shape and 
size, varying from 0.1 to 1.0 ju; conidiophores filiform, hyaline, 
continuous, 15-20 ju long, often indistinguishable from B spores. 
A and B spores present, A spores oblong-elliptical, hyaline, guttu- 
late, 5-7X1.7-3.5 m; -S spores filiform, usually hook-shaped, 
hyaline, continuous, 17-24.5X2-3.5 /x. 

Habitat. — Parasitic on kernels of Brazil nuts. 



1921] SPENCER— BRAZIL NUTS 281 

sionally four or five conidia are connected in this way (fig. 47), 
resulting, as is clearly shown by drop cultures, from germination 
succeeded by anastomosis (fig. 48). In all cultures the dense 
mycelium collects and retains water enough to germinate the 1- and 
2-celled spores, and their germ tubes anastomose readily with the 
first cells, conidial or hyphal, with which they come in contact. 
It is often difficult to distinguish between conidiophores bearing 
conidia and conidia anastomosed to hyphal cells with a short germ 
tube (fig. 44). 

Taxonomy. — The fungus is a species of Fusarium which, 
according to Wollenweber's (34) scheme of classification, belongs 
to the section Eupionnotes: chlamydospores present; perithecia 
unknown; conidia subcylindrical, sickle-shaped; base without 
pedicel, conical; terminal chlamydospores. 

4. Aspergillus decay 

General description. — Brazil nuts attacked by Aspergillus 
may give no external indication of their internal condition except 
in the most advanced stages of the disease, when the weight of the 
nut is appreciably lowered. The kernel shrinks, often cracks 
open, and is always covered with a mass of dark brown spores. 
The odor of the diseased nut is strongly rancid with a putrid taint ; 
the taste is at first sour, later very bitter. Occasionally nuts that 
are merely discolored have this same taste. Kuhl (13) states that 
Brazil nuts affected with Aspergillus flavus Mont, are poisonous, 
and that the discoloration caused by this fungus is so slight that it 
does not prevent their being eaten. Both his observations and my 
own indicate that the disease, although present, may often escape 
notice, and that it is really far more prevalent than it appears to 
be under superficial examination. Nuts in advanced stages of the 
disease, however, occur less frequently than black crust. The 
mycelium of the fungus penetrates the tissues to the center of the 
nut, and when there is a central locule, appears as a white mold on 
the walls of the locule. When the diseased kernels crack open, a 
mass of spores fills the locular space. 

Morphology. — The mycelium consists of irregularly branched 
hyphae which are slightly constricted at the septa (fig. 31). The 
cells are 20-65 X3.5-11 M? with granular contents of a faint greenish 



282 BOTANICAL GAZETTE [November 

tint. Conidiophores varying from ten to several hundred microns 
in length arise at irregular intervals from the hyphae. The shortest 
of these have little or no filamentous part, but consist merely of 
the head and sterigmata (fig. 28). Sterigmata are also borne singly 
and in groups of from two to four on the hyphal cells (fig. 27). 
The heads of the conidiophores measure 10-20 ju in diameter, and 
the sterigmata, from two to many per head, are 10-1 2X5-7 m- 
The globular, echinulated conidia are of different shades of 
yellow, and 5-10 ju in diameter, but the predominant size is 7 fx 
(fig. 29). 

Culture characters. — On cornmeal agar the rate of growth 
varies from 0.3 to 1.0 mm. daily at room temperature, and after 
forty-eight hours the central portion of the thallus shows the 
forming spore clusters in Light-Buff. The spore masses become 
darker with age until Lemon-Chrome is finally reached. On Brazil 
nut agar the growth is very similar to that on cornmeal agar, but 
with a halo 1 to 2 mm. in width, showing the presence of an extra- 
cellular, proteolytic enzyme surrounding the thallus. The color 
of the spore mass at maturity is from Orange-Cinnamon to Mikado- 
Brown. On nut plugs the growth is rapid, and a gas with the 
odor of carbon bisulphide is evident. The color of the spore mass 
is Primrose-Yellow at first, Honey- Yellow to Tawny-Olive at 
maturity. At the end of two or three months all that remains of 
the nut plug is a mass of partially disintegrated cell walls in a mass 
of mycelium. 

The growth on autoclaved rice is vigorous, with spore masses 
forming within forty-eight hours. There is little change in the 
color of the medium except for the development of a slight greenish- 
yellow tint below the spore mass. The color of the spore mass 
changes from Oil- Yellow to Orange-Cetrine. The odor of a 30-day 
old culture is very like that of cider vinegar. The nut strip above 
the water is entirely covered with spore masses within three days, 
but only about one-fourth of it is destroyed before it becomes too 
dry to support the fungus. A luxuriant growth of mycelium arises 
from the strip in the water, and usually the strip is destroyed before 
fifteen days. 



i 9 2i] SPENCER— BRAZIL NUTS 283 

Taxonomy. — A culture of this species was sent to Charles 
Thom, and the following excerpt is taken from his reply dated 
January 29, 1920: 

The organism belongs to the general group in which we are trying to 
separate three lines, the Aspergillus oryzae-flavus line, the Aspergillus wentii 
section, and the one which has been designated by Kita as Aspergillus tamari. 
This one, from the examination today, would appear to belong to the section 
containing A. tamari. Whether it is safe to identify it under an existing 
name or not would be doubtful. 

5. Bacterial decay 

General description and morphological characters.— 
When Brazil nuts are affected by this bacterial decay, the shell 
is black and greasy, and usually exhales a rancid odor. When the 
shell is cracked open the remains of the kernel are found as a 
white mass which ordinarily fills only a small portion of the shell 
cavity. Microscopic examination of fragments of the refuse shows 
numerous bacterial spores, but usually no vegetative forms and no 
fungi. When dilution plates were made from the decayed residue, 
one spore-bearing organism largely predominated. 

The vegetative cells of the organisms in cornmeal broth are 
rod-shaped, rounded at the ends, vigorously motile, and usually 
single but often in chains of from two to six individuals. The rods 
measure 2.5-5.0 juXo.8-1. 2 ju. Spores are formed within forty- 
eight hours in one end of the vegetative cells. When the cells are 
stained by Loeffler's method, the organism is found to have 
numerous long, peritrichiate flagella; stained with Loeffler's 
methylene blue the protoplasm is seen to be granular with from two 
to four darkened patches which are unevenly distributed, usually 
giving a banded effect, although often the bands are oblique as well 
as horizontal (fig. 38). The organism stains readily with methylene 
blue, Gentian violet, and carbol-fuchsin, but it is Gram negative. 

When sterile nut plugs were inoculated with the bacillus from 
pure culture, they were reduced in about fifteen days to an oily 
mass which, in all essential characters, was like the remains of the 
nut kernels in the natural cases of nut decay. Dilution plates 
made from nut meats that had decayed, following pure culture 



284 BOTANICAL GAZETTE [November 

inoculation, showed only one type of colony, and this proved to 
consist of the organism with which the plugs had been inoculated. 
The organism had no appreciable effect on the nut strip above the 
water, and the strip in the water was only very slowly decomposed, 
but strips in cornmeal bouillon were completely destroyed within 
ten to fifteen days. The organism grows best in the presence of 
air, as the colonies on all plated media and stab culture show, but 
deep lenticular colonies (fig. 40), and colonies next to the glass 
(fig. 39) in agar plates, as well as the faint line of growth along 
the stab, indicate that it is a facultative anaerobe. 

While none of the usual tests for particular enzymes was 
made, the reactions in different culture media indicate the produc- 
tion of diastase, invertase, rennet, and pepsin. In Brazil nut agar 
plates there is formed a transparent halo about the colony, and as 
the opacity of the agar is due to the presence of solid proteid matter 
(20), the halo results from the digesting of these proteids. There 
is an abundant secretion of the protease which makes the halo, 
as the diameter of the transparent area is from two to three times 
that of the colony itself. This enzyme was precipitated as already 
described, and drops of a water solution of the dried precipitate 
placed on Brazil nut agar plates. A transparent area as large as 
the drop of solution was formed in a plate 2 mm. thick in from two 
to three hours. 

The organism seems to be an undescribed one, and a complete 
description of it will be given in a separate paper. 

6. Actinomyces decay 

General description and morphology. — Empty shells that 
are intact and still retain their normal color are occasionally found 
among Brazil nuts. When these shells are cracked open a char- 
acteristic musty odor is evident, and the inner shell wall is seen 
to be covered with pinkish velvety pustules that are from one to 
several millimeters in diameter. Water mounts of pieces of a 
pustule show tenuous, mycelial-like strands, or chains of spores 
which readily stain with carbol-fuchsin. The filaments are not 
long but branch, and the mass is so bound together by the branches 
that it is quite impossible to separate entire filaments from the mass. 



ig2i] SPENCER—BRAZIL NUTS 285 

The filaments are never entirely straight nor yet very crooked, and 
chains of spores are usually contained in the free ends (fig. 37). 
No spirals were found on any of the media. The diameter of the 
filaments varies from 1.0 to 1.3 /z, and the oblong spores measure 
1.6X0.8 fX. 

The germination of spores was studied with an oil immersion 
lens, in a hanging drop prepared as follows. A thin film of synthetic 
agar was spread on a thin cover-glass, and a loop full of a dilute 
spore suspension placed on the agar film. This was inverted over 
a dry Van Tieghem cell. The water soon evaporated, leaving the 
spores in contact with the agar, where their germination was 
easily studied and camera lucida drawings made. According to 
Drechsler (9), Actinomyces spores produce from one to four 
germ tubes, "the approximate number being more or less char- 
acteristic of the species." This species produces one and two 
germ tubes which often branch directly on leaving the conidium 

(% 36). 

The organism was studied in the manner suggested by Conn 
(3) and Waksman (33), and the media were made in accordance 
with directions given by Waksman (33). The following culture 
characters were noted: 

Cultural characters. — 1. Synthetic agar: room tempera- 
ture, after ten days: growth densely compact but thalli small, at 
first white, but after ten days Pale Pinkish Buff; aerial mycelium 
white and dense; soluble pigment none. 

2. Calcium malate-glycerin agar: growth spreading and not 
zonated, bordered by submerged mycelial bands of varying width, 
pearl white ; aerial mycelium short, loose, and pearl white ; soluble 
pigment. 

3. Glucose agar: growth luxuriant, color same as in synthetic 
agar, thallus conspicuously zonated; aerial mycelium white to 
Pale Pinkish Buff, powdery; soluble pigment none. 

4. Glycerin agar: growth densely compact, not zonated, Pale 
Pinkish Buff; aerial mycelium powdery, white; soluble pigment 
none. 

5. Brazil nut agar: growth rapid, densely compact with wide 
margin of submerged mycelium, white to Pale Pinkish Buff; 



286 BOTANICAL GAZETTE November 

aerial mycelium dense, white; soluble pigment none; enzymatic 
zone three to four times the diameter of thallus. 

6. Cornmeal agar: growth dense but zonated, Pale Pinkish 
Buff; aerial mycelium powdery; soluble pigment none. 

7. Egg albumin agar: growth thin, conspicuously zonated, 
Pale Pinkish Buff; aerial mycelium powdery, Pale Pinkish Buff; 
soluble pigment none. 

8. Nut plugs: growth vigorous, Pale Pinkish Buff; aerial 
mycelium powdery, white; medium not completely destroyed, 
but much shrunken and blackened. 

9. Autoclaved rice: growth vigorous, Pale Pinkish Buff; aerial 
mycelium 2 cm. ; almost completely destroyed in sixty days. 

10. Potato plugs: growth vigorous, crumpled, Pale Pinkish 
Buff; aerial mycelium abundant, at first white, later Pale Pinkish 
Buff; medium with no change in color, much reduced in size in 
two months. 

11. Carrot plugs: growth at first slow, appearing after five 
days, crumpled and dense, Pale Pinkish Buff; aerial mycelium 
powdery, at first white, later Pale Pinkish Buff; medium darkened 
near the growth, no change in color in other regions, much shrunken. 

12. Brazil nut bouillon: growth surface pellicle, snow-white; 
aerial mycelium white, powdery; medium somewhat clarified. 

13. Nut strips: growth slight on strip above water, and none on 
strip in water; no growth on surface of water. 

Biochemical features. — The proteolytic enzyme which makes 
the halo in Brazil nut agar plates was the only one studied, but the 
growth reactions in different media were taken to indicate the 
probable production of several other enzymes, diastase and inver- 
tase especially. The proteolytic enzyme was isolated by precipita- 
tion, as previously described, and its proteolytic power tested by 
placing drops of a water solution of the dried precipitate on Brazil 
nut agar plates. Transparent areas the size of the drops developed 
in from two to three hours, depending upon the thickness of the 
agar plates. 

Taxonomy. — The organism is an Actinomyces which, according 
to Waksman's key, belongs in division B, "no soluble pigment 
produced on gelatin or other protein media," and in section /, 



1921] SPENCER— BRAZIL NUTS 287 

"species strongly proteolytic; gelatin liquefied rapidly, milk 
clotted and peptonized rapidly." No species given in this division 
and section, however, has the characteristics of the one found in 
Brazil nut shells. It is therefore given the name of Actinomyces 
brasiliensis . 

Actinomyces brasiliensis, n. sp. — -Straight, branched hyphae 
1. 0-1.3 M i n diameter; spores borne in chains in free ends of hyphae, 
oblong, 1.6X0.8 n; growth Pale Pinkish Buff on all agars except 
calcium malate-glycerin, on which it is white; zonated on glucose, 
cornmeal, and egg albumin agars; aerial mycelium on all media, 
white to Pale Pinkish Buff; no soluble pigment formed. 

Habitat. — Parasitic on kernels of Brazil nuts. 

7. Phomopsis decay 

General description. — Only one nut was found affected 
with Phomopsis decay, but because of its striking diagnostic 
features the fungus was isolated and studied. There was no 
external indication of the diseased condition, but the kernel of the 
nut was rich brown, with a few black specks near one end. The 
odor of the nut was pleasant and the taste agreeable. Stained 
hand sections showed that the mycelium of the fungus had pene- 
trated into the radicle to considerable depth. 

Morphology. — The mycelium was tenuous, septate, and at 
first hyaline, but soon became brown or smoke colored. According 
to Diedicke (8), the form of the pycnidia is greatly varied. In the 
Brazil nut species several of the forms pictured by Diedicke were 
observed, but the one most commonly met with was mammiform, 
with a wartlike protuberance. The irregular pycnidial cavity so 
common to the genus was frequently observed, but a regular 
cavity was the rule. Two forms of spores were present in all 
pycnidia examined (fig. 50), and as is customary, the Phoma type 
will be designated as A, the filamentous as B spores. The B 
form did not germinate in hanging drop, a fact supporting the 
statement made by Grove (10) that these may or may not be 
spores. When they fail to germinate they are probably what 
Saccardo (25) took them to be, conidiophores, which according 
to Grove "become more curved than when in situ." The A 



288 BOTANICAL GAZETTE [November 

spores are oblong-elliptical, hyaline, guttulate, and measure 
5-7X1.7-3.5 m- The B spores are filiform, usually hook-shaped, 
hyaline, continuous, and measure from 17-24.5X2-3.5 /x. 

Culture characters. — The fungus grew well on all media 
used. On cornmeal agar the thallus was circular and without 
zonations. A loose aerial mycelium covered the entire thallus, 
and numerous pycnidia varying in size were scattered Over the 
surface of the plate. The pycnidia appeared simultaneously with 
the brown color, which was usually noticed after five or six days. 
On Brazil nut agar the growth was similar to that on cornmeal 
agar. The clear halo formed in the agar plate barely exceeded 
the size of the thallus. On autoclaved rice a brown or smoky color, 
due to the mycelial growth, was noticeable, but no color change 
occurred in the medium. Nut plugs were soon covered with a 
brown mycelium which later became almost black. The surface 
was soon covered with black, wartlike pycnidia, and the entire 
mass when cut through suggested a dried sponge. The odor was 
similar to that of very rancid nuts. The fungus made no growth 
on nut strips above the water, but a dense mass of mycelium, filled 
with black strands, developed on the strips in the water. These 
strips retained their form, but the cessation of mycelial growth, 
which occurred between ten and fifteen days after inoculation, 
marked the time of nutrient exhaustion. 

Taxonomy. — The fungus is a typical Pliomopsis which 
approaches P. aucubicola Grove more nearly than any other 
described species. A spores are shorter, and this, coupled with the 
fact that it occurs on an unrelated host, necessitates describing it 
as new. 

Phomopsis bertholletianum, n. sp. — Pycnidium dark brown, 
mammiform, with wartlike protuberance, irregular in shape and 
size, varying from 0.1 to 1.0 ax; conidiophores filiform, hyaline, 
continuous, 15-20 ax long, often indistinguishable from B spores. 
A and B spores present, A spores oblong-elliptical, hyaline, guttu- 
late, 5-7X1.7-3.5 m; B spores filiform, usually hook-shaped, 
hyaline, continuous, 17-24.5X2-3.5 ax. 

Habitat. — Parasitic on kernels of Brazil nuts. 



1921] SPENCER— BRAZIL NUTS 289 

8. Bitter rot 

Figure 4 shows a part of a Brazil nut affected with bitter rot, and 
fig. 49 shows spores of the fungus, two of which have conidiophores 
attached. Neither the spores nor the mycelium was viable, and 
time prevented more than a superficial examination being made. 
The fungus is apparently a Myxosporium. 

State Teachers College 
Cape Girardeau, Mo. 

LITERATURE CITED 

1. Berg, Otto, Bertholletia excelsa Hb. et Bpl., Martii Flora Braziliensis. 
14:478. 1859. 

2. Chamberlain, C. J., Methods in plant histology. University of Chicago 
Press. Chicago. 191 5. 

3. Conn, H. J., The possible function of Actinomyces in soil. Jour. Bact. 
1:197. 1916. 

4. , Soil flora studies, V. Actinomyces in soil. N.Y. Agric. Expt. 

Sta. Tech. Bull. 60. 1917. 

5. Chester, F. D., A manual of determinative bacteriology. Macmillan 
Co. New York. 1914. 

6. Corbet, L. C, Cold storage. W.Va. Agric. Expt. Sta. Bull. 75. 1901. 

7. Department of Commerce. Brazil and Cream nuts. Monthly Summary 
of Foreign Commerce of the United States. December, 1919. 

8. Diedicke, H., Die Gattung Phomopsis. Ann. Myc. 9:8. 191 1. 

9. Drechsler, C, Morphology of the genus Actinomyces. Bot. Gaz. 67 :65~ 
&3; 147-168. 1919. 

10. Grove, W. B., The British species of Phomopsis. Roy. Bot. Gard., Kew 

Bull. Misc. Inform, no. 2. 1917 (p. 49). 
n. Higgins, B. B., Plum wilt, its nature and cause. Ga. Agric. Expt. Sta. 

Bull. 118. 1916. 

12. Jones, L. R., Pectinase the cytolytic enzyme produced by Bacillus caro- 
tovorus and certain other soft-rot organisms. N.Y. Agric. Expt. Sta. 
Tech. Bull. 11. 1909. 

13. Kuhl, Hugo, Uber eine eigenartige Yeranderung der Paranuss. Pharm. 
Zehtralh. 51 :io6. 1910. 

14. Lindau, G., Trichothecium roseum. Rabenhorst's Kryptogamen-Flora. 
8:366. 1907. 

15. McAlpine, D., Fungus diseases of stone-fruit trees in Australia. Dept. 
Agric, Melbourne, Victoria. 1902. 

16. McMurran, S. M., Diseases of trees. Amer. Nut Jour. 4:81. 1916. 



290 BOTANICAL GAZETTE [November 

17. Mangin, M. L., La Pourriture des Chataignes. L'Aced. D'Agr. France. 
4:885. 1918. 

18. Martz, J., Diseases and insect pests of the pecan. Fla. Agric. Expt. Sta. 
Bull. 147. 1918. 

19. Migula, W., System der Bacterien. Handbuch der Morphologie Ent- 
wickelungsgeschichte und Systematik der Bacterien. Verlag von Gustav 
Fischer, Jena. 1900. 

20. Osborne, T. B., and Clapp, S. H., Hydrolysis of excelsin. Amer. Jour. 
Phys. 19:53- 1907- 

21. Pierce, N. B., Walnut bacteriosis. Bot. Gaz. 31 1272. 1901. 

22. Pearson, C. H., Uses of the Brazil nut tree. Amer. Forestry. 23:782. 
1919. 

23. Rand, F. V., Some diseases of pecans. Jour. Agric. Res. 1:303. 1914. 

24. Rumbold, Caroline, Notes on chestnut fruits infected with the chestnut 
blight fungus. Phytopath. 5:64. 1915. 

25. Saccardo, P. A., Sylloge Fungorum. Vol. XVIII. 

26. Shear, C. L., and Stevens, Neil E., Cultural characters of the chestnut 
blight fungus. U.S. Dept. Agric. B.P.I. Cir. 131. 1913. 

27. Smith, R. E., Walnut culture in California, walnut blight. Cal. Agric. 
Expt. Sta. Bull. 231. 191 2. 

28. Strasburger, E., Textbook of botany, 4th Eng. ed. Macmillan Co. 
London. 191 2. 

29. Stuckey, R. P., Pecans, varieties, influence of climate, soil, stock on scion. 
Ga. Agric. Expt. Sta. Bull. 116. 1915. 

30. Turner, William F., Nezara viridula and kernel spot of pecan. Science 
47: 490. 1918. 

31. Von Ivanoff, K. S.. Uber Trichotheciiim roseum Link als Ursache der 
Bitterfaule von Frvcten. Zeitsch. PfTkheit 14:36. 1904. 

32. Vaughan, R. E., A method for the differential staining of fungus and host 
cells. Ann. Mo. Bot. Gard. 1:241. 1914. 

33. Waksman, S. A., Cultural studies of species of Actinomyces. Soil Sci. 
8:71. 1918. 

34. Wollenweber, Ff. W., Ramularis, Mycosphaerella, Nectria, Calonectria. 
Eine Morphologisch Pathologische Studie zur Abgrenzung von pilzgruppen 
mit cylindrischen und sichelformigen Konidienformen. Phytopath. 3: 
197. 1913. 

35. Young, W. J., A study of nuts with special reference to microscopic identi- 
fication. U.S. Dept. Agric. Bur. Chem. Bull. 160. 

EXPLANATION OF PLATES VIII-XII 

All drawings were made with camera lucida. 

PLATE VIII 

Fig. 4. — Part of Brazil nut kernel affected by bitter rot; Xi. 

Fig. 5. — Thalli of Actinomyces brasiliensis n. sp. on cornmeal agar; Xf. 



1921] SPENCER—BRAZIL NUTS 



291 



Fig. 6. — Thallus of Actinomyces brasiliensis n. sp. on cornmeal agar; X25. 

Fig. 7. — Colony of Bacillus from Brazil nut on cornmeal agar; X2. 

Fig. 8. — Pellioniella macrospora n. sp. in autoclaved rice; tube on right 
20 days old; tube on left 10 days old; Xi. 

Fig. 9.— Brazil nut 30 days after inoculation with Pellioniella macrospora 
n. sp. ; nut plug at top marks place of inoculation, and along two edges inner 
seed coat removed to expose blackened endosperm ; X 1 . 

Fig. 10. — Thallus of Actinomyces brasiliensis n. sp. on Brazil nut agar, sur- 
rounded by transparent area in which proteids have been digested owing to 
secretion of proteolytic enzyme; XiJ- 

Fig. 11. — Thallus of Pellioniella macrospora n. sp. on cornmeal agar: 
three zones: (1) scarcely visible outer zone of white; (2) zone of nearly same 
width that is green in growing thallus; (3) inner black circle; X|. 

Fig. 12. — Actinomyces brasiliensis n. sp. on potato plug after 10 days; Xi. 

PLATE IX 

Fig. 13. — Paraphyses, immature conidia, and conidiophores of Pellioniella 
macrospora n. sp.; X500. 

Fig. 14. — Conidia-like cells of hyphae of P. macrospora n. sp. from dis- 
eased tissue of Brazil nut; X500. 

Fig. 15. — Mature, immature, and transitional stages in development of 
conidia of P. macrospora n. sp. ; X500. 

Fig. 16. — Germination of immature conidia of P. macrospora n. sp., planted 
in same hanging drop with mature conidia shown in figure 18; X500. 

Fig. 17. — Germination of mature conidia of P. macrospora n. sp.; germ 
tubes from one to two hours longer in emerging than those of immature conidia 
shown in figure 17; X 500. 

Fig. 18. — Hypha of P. macrospora n. sp., showing most common type of 
cell; X500. 

Fig. 19. — Hypha of P. macrospora n. sp. from near pycnidium; X500. 

Fig. 20. — -Conidia-like cells of hyphae of P. macrospora n. sp., taken from 
culture in autoclaved rice; X500. 

Fig. 21. — Section of pycnidium of P. macrospora n. sp., enlarged about 
450 diameters. 

Fig. 22. — Hyphae of P. macrospora n. sp., showing two types of cells; 
X500. 

PLATE X 

Fig. 23. — Conidia of Cephalosporium bertholletianum n. sp.; X500. 

Fig. 24. — Hyphae, conidiophores, and spore masses surrounded by water 
drops, C. bertholletianum n. sp.; X500. 

Fig. 25. — Germinating conidia of C. bertholletianum n. sp., two hours after 
planting; X500. 

Fig. 26. — Germinating conidia of C. bertholletianum n. sp., twenty hours 
after planting; X500. 



292 BOTANICAL GAZETTE [November 

Fig. 27. — Hyphae of Brazil nut Aspergillus bearing sterigmata ; X500. 

Fig. 28. — Hypha of Brazil nut Aspergillus bearing short stalked conidio- 
phores; X500. 

Fig. 29. — Conidia of Brazil nut Aspergillus; X500. 

Fig. 30. — Mature conidiophores of Brazil nut Aspergillus; X500. 

Fig. 31. — Hyphae showing branching habit and anastomosis, Brazil nut 
Aspergillus; X500. 

Fig. 32. — Early stages of conidiophores of Brazil nut Aspergillus; X500. 

PLATE XI 

Fig. 33. — Microtome section of normal nut kernel showing tissues named 
in order, beginning at top: endosperm, epidermis, cortex, procambium, and 
medulla; X500. 

Fig. 34. — Microtome section of Brazil nut affected by Pellioniella macro- 
spora n. sp., showing dense mycelial growth in endosperm region; Xiooo. 

Fig. 35. — Microtome section of Brazil nut affected by P. macrospora n. sp., 
showing relation of fungus to host tissues; X500. 

Fig. 36. — Germinating conidia of Actinomyces brasiliensis n. sp.; Xiooo. 

Fig. 37. — Hyphae and conidia, Actinomyces brasiliensis n. sp.; Xiooo. 

Fig. 38. — Vegetative cells of Brazil nut bacillus; Xiooo. 

Fig. 39. — Colony of Brazil nut bacillus growing near glass in cornmeal 
agar plate; X50. 

Fig. 40. — Deep colony of Brazil nut bacillus n. sp.; X50. 

Fig. 41. — Surface colony of Brazil nut bacillus n. sp.; X50. 

PLATE XII 

Figs. 42-49, and 51 are of a Fusarium which causes dry rot of Brazil nuts. 

Fig. 42. — Conidial variation and anastomosis; X500. 

Fig. 43. — Typical hyphae showing difference in size and branching habit; 
X500. 

Fig. 44. — Hyphae bearing single conidiophores and conidia anastomosed 
to hyphal cells by germ tube; X500. 

Fig. 45. — Terminal chlamydospores; X500. 

Fig. 46. — Germinating conidia in hanging drop culture; X500. 

Fig. 47. — Anastomosing conidia and hyphae from culture plates; X500. 

Fig. 48. — Anastomosis of germinating conidia in hanging drop; X500. 

Fig. 49. — Conidia and conidiophores of bitter rot fungus, taken from 
pustules on diseased kernel ; X 500. 

Fig. 50. — Phomopsis conidia, two having germ tubes attached; X500. 

Fig. 51. — Hyphae of Fusarium from washed agar plates; X500. 



BOTANICAL GAZETTE, LXXII 



PLATE VIII 










f | 








^%ld^ / ^'^um-^' 




SPENCER on BRAZIL NUTS 



BOTAXICAL GAZETTE, LXXII 



PLATE IX 




SPENCER on BRAZIL NUTS 



BOTANICAL GAZETTE, LXXII 



PLATE X 




SPENCER on BRAZIL NUTS 



BOTAXICAL GAZETTE, LXXI1 



PLATE XI 




39 40 

SPENCER on BRAZIL NUTS 



BOTANICAL GAZETTE, LXXII 



PLATE XII 




/ T'rf Hi 




ACKNOWLEDGMENT 

The writer wishes to acknowledge his indebtedness to Dr. F. L. Stevens, 
professor of plant pathology, University of Illinois, under whose direction these 
studies were conducted; to Professor William Trelease, head of the depart- 
ment of botany, University of Illinois, for suggestions relating to taxonomy; 
to Professor Fred W. Tanner, for assistance rendered in connection with the 
bacterial investigations; and to Grossfeld & Roe Company, Hills Brothers 
Company, and Kelling-Karel Company of Chicago, for samples of nuts and 
information relating to the commercial aspect of the problem. 



VITA 

The author of this thesis was born near Rushville, Schuyler County, 
Illinois, February 13, 1881. He taught school in his home county from 1901 
to 1907 when he entered the University of Illinois. In 191 1 he received the 
A.B. degree and became supervisor of schools at Waverly, Illinois. In 1912 
he returned to the university on a scholarship in education, but at the end of 
the first semester resigned the scholarship to accept an assistantship in 
psychology. This prevented his finishing his thesis on "Defoe's Contribu- 
tions to Education," but during the next year, while supervising a township 
high school, the thesis was finished and he received the M.A. degree in June, 
1914. In 191 7 he returned to the university as an assistant in botany, and 
received the degree of Doctor of Philosophy in June, 1920. 



